Diabetes mellitus is a common degenerative disease affecting 5 to 10 percent of the population in developed countries. It is a heterogenous disorder with a strong genetic component; monozygotic twins are highly concordant and there is a high incidence of the disease among first degree relatives of affected individuals. The propensity for developing diabetes mellitus is reportedly maternally inherited, suggesting a mitochondrial genetic involvement. Alcolado, J. C. and Alcolado, R., Br. Med. J. 302: 1178-1180 (1991); Reny., S. L., International J. Epidem. 23: 886-890 (1994).
Studies have shown that diabetes mellitus may be preceded by or associated with certain related disorders. For example, it is estimated that forty million individuals in the U.S. suffer from late onset impaired glucose tolerance (IGT). IGT patients fail to respond to glucose with increased insulin secretion. A small percentage of IGT individuals (5-10%) progress to insulin deficient non-insulin dependent diabetes (NIDDM) each year. Some of these individuals further progress to insulin dependent diabetes mellitus (IDDM). This form of NIDDM or IDDM is associated with decreased release of insulin by pancreatic beta cells and/or a decreased end-organ response to insulin. Other symptoms of diabetes mellitus and conditions that precede or are associated with diabetes mellitus include: obesity, vascular pathologies, peripheral and sensory neuropathies, blindness, and deafness.
Due to the strong genetic component of diabetes mellitus, the nuclear genome has been the main focus of the search for causative genetic mutations. However, despite intense effort, nuclear genes that segregate with diabetes mellitus are known only for rare mutations in the insulin gene, the insulin receptor gene, the adenosine deaminase gene and the glucokinase gene.
The maternal heredity associated with diabetes mellitus suggests that mitochondrial inheritance might play a role, since mitochondrial genes are maternally inherited. Indeed, a rare form of late-onset NIDDM associated with nerve deafness appears to segregate with a point mutation in a mitochondrial tRNA gene (tRNA.sup.leu). Individuals carrying this mutation often present with impaired insulin secretion in response to glucose and are usually given the diagnosis of insulin dependent diabetes mellitus (IDDM), slowly progressive IDDM, or insulin deficient non-insulin dependent diabetes (NIDDM). Although this mutation accounts for less than 1% of NIDDM cases, it raises the possibility that other mutations in mtDNA may associate with NIDDM.
Mitochondrial DNA (mtDNA) is a small circular DNA that is approximately 17 Kb long in humans. The mtDNA encodes for two ribosomal RNAs (rRNA), a complete set of transfer RNAs (tRNA), and thirteen proteins, including two ATP synthase genes, ATP synthase subunits 6 and 8.
Most of the mtDNA present in an individual is maternally derived from the mtDNA contained within the ovum at the time of the individual's conception. Mutations in mtDNA sequences that affect all copies of mtDNA in an individual are known as homoplasmic. Mutations which affect only some copies of mtDNA are known as heteroplasmic and will vary between different mitochondria in the same individual.
Despite indications of a possible mitochondrial etiology for at least some forms of diabetes mellitus, neither the incidence nor the exact mechanism producing mitochondrial transport dysfunction in late onset diabetes is known, nor has a genetic or structural basis for these dysfunctions been identified. Without knowing what causes these electron transport dysfunctions and in particular the genetic or structural basis, it is difficult to diagnose or treat late onset diabetes.
Clearly then, a reliable diagnosis of late onset diabetes at its earliest stages is critical for efficient and effective intercession and treatment of this debilitating disease. There is a need for a non-invasive diagnostic assay that is reliable at or before the earliest manifestations of late onset diabetes symptoms. There is also a need for developing therapeutic regimens or drugs for treating both the symptoms of diabetes mellitus and the disease itself.
However, the identification of diagnostic assays and of therapeutic regimens or drugs that are useful in the treatment of disorders associated with mitochondrial defects has historically been hampered by the lack of reliable model systems that could be used in rapid and informative screening. Animal models do not exist for many of the diseases that are associated with mitochondrial gene defects. Appropriate cell culture model systems are either not available, or are difficult to establish and maintain. Furthermore, even when cell culture models are available, it is often not possible to discern whether the mitochondrial or the nuclear genome is responsible for a given phenotype, as mitochondrial functions are often encoded by both nuclear and mitochondrial genes. It is, therefore, also not possible to tell whether the apparent effect of a given drug or treatment operates at the level of the mitochondrial genome or elsewhere.
One approach that has been useful in discerning which genome is responsible is to eliminate the mitochondrial DNA in cultured cells known to have proper mitochondrial function and then transfer to such cells the mitochondria from diseased patients. However, the resulting cell lines, called .rho..degree. cell lines, tend to be unstable and hard to culture. Fully differentiated cell lines are used as the targets for transplantation, but their naturally limited life spans makes them particularly unsuitable for screening purposes. In addition, such transformations have not been done using cells of the type that are most affected by the disease, making it unclear whether the mitochondrial deficiencies observed in the transformants are related to the disease state being studied.
The present invention satisfies these needs for a useful diagnostic and effective treatment of late onset diabetes and provides related advantages, as well.